Opioid derivative

ABSTRACT

1. A peptide derivative represented by the following formula (1) or a salt thereof;  
                 
 
     , wherein R 1  is hydrogen atom or methyl group, R 2  is hydrogen atom or hydroxy group and n is an integer of 1-8, provided that R 1  is hydrogen atom when R 2  is hydrogen atom, which has specific and high binding affinity with the μ-opioid receptor.

TECHNICAL FIELD

[0001] The present invention relates to a new peptide derivative or itssalt which has excellent physiological activity via the opioid receptor,especially shows binding affinity to the opioid receptor in lowerconcentration. Further the present invention relates to a medicalcomposition containing said peptide as an active ingredient and itsmedical use.

PRIOR ART

[0002] It is known that peptides show various physiological activitiesby changing their amino acid sequence. A peptide mimetic prepared bychanging a peptide-component, an amino acid with another amino acid orother substituent, tends to show another specific activity. Many studieson these peptides, generally called opioid peptides such as enkephalin,etc., which are present in brain and participate in analgesic activityetc., have been carried out, as well as with opioid mimetics, which aresynthetic opioid peptides resembling naturally occurring compounds.

[0003] It is reported that expression of analgesic activity occurs byinteraction between ligand and receptor.

[0004] Morphine has been known as an opioid-receptor activatingsubstance, and opioid type analgesics, morphine homologues have beenclinically used for inhibition of cancer pain, acute pain afterskin-grafting or post varicella-zoster.

[0005] However, these analgesics show severe side effects such ascentral nervous system depressive activity, leiomyotention of digestivetract, addiction, toleration etc. and therefore, further improvedanalgesics, in many fields especially such as anaphase of cancer, painafter post varicella-zoster are strongly desired.

DETAILED DESCRIPTION OF INVENTION

[0006] The present inventors have recently invented to form a pyrazinonering from dipeptidyl chloromethyl ketone and have succeeded in preparingopioid mimetics therefrom (Okada et al., Tetrahedron Lett. 55,14391-14406 (1999)). Some compounds among them were found to be weak inactivity but to bind specifically with the μ-opioid receptor.

[0007] Furthermore, the present inventors have studied with expectationfor preparing more selective opioid mimetic analgesics with higheractivity using the simple alkyl chain instead of said pyrazinone ring.Namely, the present inventors planed to introduce tyrosine,phenylalanine or 2,6-dimethyltyrosine (Salvadori et al., MolecularMedicine, 1, 678-689(1995) at the both amino groups of alkanediamine(NH₂-alkyl-NH₂) for the purpose to enhance specific and high bindingaffinity toward the μ-opioid receptor.

[0008] As a result, it has been found that peptide derivativesrepresented by the following formula (1) have specific and high bindingaffinity with the μ-opioid receptor and show different analgesicactivity from morphine, and the present invention has been completed.

[0009] The present invention relates to a peptide derivative representedby the following formula (1) or a salt thereof;

[0010] , wherein R¹ is hydrogen atom or methyl group, R² is hydrogenatom or hydroxy group and n is an integer of 1-8, provided that R¹ ishydrogen atom when R² is hydrogen atom, which shows specific and highbinding affinity with the μ-opioid receptor.

[0011] Further, the present invention relates to a pharmaceuticalcomposition containing at least one peptide derivative represented bythe formula (1) or one salt thereof as an active ingredient.

[0012] Further, the present invention relates to a μ-opioid receptoragonist activator containing at least one peptide derivative representedby the formula (1) or one salt thereof as an active ingredient.

[0013] Further, the present invention relates to a method for inhibitingor moderating pain by administering at least one peptide derivativerepresented by the formula (1) or one salt thereof having μ-opioidreceptor agonist activation activity as an active ingredient in aneffective dosage to a patient having pain or suffering from nervousdefect disease (neuropathy) related to μ-opioid receptor activation.

EMBODIMENT OF INVENTION

[0014] The peptide derivative in the present invention consists ofbinding 2,6-dimethyltyrosine (abbreviated as Dmt) at one amino group ofdiaminoalkane, and binding tyrosine, phenylalanine, or2,6-dimethyltyrosine at the other amino group of the diaminoalkane, andall the amino acids are levorotary.

[0015] The peptide derivative represented by the formula (1) is easilyprepared by the liquid phase method.

[0016] For example, a peptide is prepared by coupling 1,4-dimethylalkanewith Boc-Dmt-OH by BOP(benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate),and thus prepared peptide is subject to TFA-treatment and hydrochloricacid-treatment in dioxane to give a peptide derivative (1) wherein R¹ ismethyl group and R² is hydroxy group.

[0017] The synthesis scheme of the above compound, e.g. thehydrochloride is shown below.

[0018] , wherein n is an integer of 1-8.

[0019] The synthesis scheme of the opioid derivatives (1) of the presentinvention, wherein R¹ is hydrogen atom and R² is hydrogen atom orhydroxy group and n is an integer of 4 is shown below.

[0020] , wherein R² is hydrogen atom or hydroxy group.

[0021] In the method for preparation of the peptide derivative (1) ofthe present invention, the objective peptide derivative and anintermediate thereof are isolated and purified by being appropriatelycombined with various methods such as ion chromatography, gelchromatography, reverse phase chromatography, recrystallization,extraction, etc.

[0022] Thus obtained peptide derivative (1) of the present invention canbe converted into a salt thereof using an organic or inorganic acidaccording to the conventional method.

[0023] The preferable compound of the present invention is a peptidederivative (1) of the present invention wherein n is an integer of 4 to6, and further preferably R² is hydroxy group and furthermore preferablyR¹ is methyl group.

[0024] The preferable salt is a pharmaceutically acceptable salt, suchas hydrochloride or methanesulfonate.

[0025] The starting material, dimethyltyrosine can be prepared inaccordance with a method described in Dygos et al. (Synthesis, 741(1991)).

[0026] The peptide derivative (1) of the present invention or a saltthereof is not described in any publication, and has specific and highbinding affinity with the μ-opioid receptor and exhibits morphinelike-physiological activity, such as analgesic activity.

[0027] Therefore, the peptide derivative (1) of the present invention ora salt thereof exhibits analgesic activity and the central nervoussystem or peripheral nervous system response which the opioid peptideexpresses via its receptor, such as physiological activities, e.g.,anesthesia, pulse, digestion mechanism, hormone releasing control,cardiac muscle contraction control.

[0028] The peptide derivative (1) of the present invention or a saltthereof can be used as analgesics and as a therapeutic or prophylacticdrug for diseases with the nervous system defect related to other opioidreceptor activation.

[0029] The peptide derivative (1) of the present invention or a saltthereof is orally, paraorally, intrarectally, sublingually or topicallyadministered, or is administered by epidural injection in the spinalcord or by intra-arachinoid injection. The peptide derivative (1) of thepresent invention or a salt thereof can be combined with otheringredients suitable for the purposed preparation (form) to beadministered to the patient.

[0030] Dosage of the peptide derivative (1) of the present invention ora salt thereof depends on age, degree of diseases etc., but is usually0.01-500 mg/kg/day and may thus be divided.

[0031] The following abbreviations used in the present specificationmean as follows;

[0032] AcOEt: Ethyl acetate

[0033] Boc: tert-Butoxycarbonyl

[0034] BOP: Benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate

[0035] BrZ: 2-Bromobenzyloxycarbonyl

[0036] DIEA: Diisopropylethylamine

[0037] DMF: Dimethylformamide

[0038] Dmt: 2,6-dimethyl-L-tyrosine

[0039] Fmoc: 9-Fluorenylmethoxycarbonyl

[0040] MeCN: Acetonitrile

[0041] PyBOP: Benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate

[0042] Et₃N: Triethylamine

[0043] TFA: Trifluoroacetic acid

[0044] HEPES: N-2-Hydroxyethylpiperazine-N′-2-ethane sulfonic acid

[0045] GDP: Guanosine 5′-diphosphate

[0046] DAGO: H-Tyr-D-Ala-Gly-MePhe-Gly-ol

[0047] DPDPE: H-Tyr-cyclo(D-Pen-Gly-Phe-D-Pen)

[0048] The present invention is illustrated by the following examples,reference examples and experimental tests, but is not limited by them.

EXAMPLE

[0049] General Procedure

[0050] Melting points were determined on a Yanagimoto micro-meltingpoint apparatus without correction.

[0051] Optical rotations were measured with an automatic polarimeter,model DIP-360 (Japan Spectroscopic Co.).

[0052] Matrix assisted laser desorption time-of-flight mass spectra(MALDI-TOF-MS) were obtained on a Kratos MALDI II mass spectrometer(Kratos Analytical).

[0053] NMR: ¹H-(500 MHz) and ¹³C-(125 MHz)NMR spectra were recorded on aBruker ARX500 spectrometer.

[0054] Chemical shifts are expressed as ppm down field fromtetramethylsilane, used as an internal standard (δ-value). The J valuesare given in Hz. The ¹³C signals were assigned with the aid ofdistortionless enhancement by polarization transfer (DEPT), and 2Dexperiments, and multiplicities are indicated by a p (primary), s(secondary), t (tertiary) or q (quaternary). Waters model 600E was usedfor analytical or preparative HPLC. Peptides were analyzed by reversephase HPLC on a COSMOSIL C18 column (4,6×250 mm, Nakarai) or YMC R&DR-ODS-5-A (4.6×250 mm).

[0055] Retention time was shown as tR (C) or tR (Y), respectively. Thecolumn was eluted by using a linear gradient from 10% acetonitrile inwater, containing 0.05% TFA by increasing 1% acetonitrile concentrationin one min. at a flow rate of 1 ml/min; Detection was at 220 nm. On TLC(Kieselgel 60G. Merck), Rf¹, Rf², Rf³, Rf⁴ and Rf⁵ values refer to thesystems of CHCl₃, MeOH and AcOH (90:8:2); CHCl₃, MeOH and H₂O (89:10:1);CHCl₃, MeOH and H₂O (8:3:1, lower phase); n-BuOH, AcOH and H₂O (4:1:5,upper phase); n-BuOH, AcOH, pyridine and H₂O (4:1:1:2), respectively.

[0056] 0.1% Ninhydrin-Aceton Solution was used for detection of aminogroup. Hydrogen bromide-Ninhydrin Method was used for detection ofBoc-protecting group.

Example 1 1) 1,4-Bis-(N^(α)-Boc-Dmt-amino)butane

[0057] BOP reagent (700 mg, 1.6 mmol) was added to a solution ofN^(α)-Boc-Dmt-OH (500 mg, 1.6 mmol), 1,4-diaminobutane (60 mg, 0.68mmol) in DMF (15 ml) containing Et₃N (0.66 ml, 4.7 mmol) at roomtemperature. The reaction was stirred for 18 hr at room temperature.After removal of the solvent, the residue was extracted with AcOEt,which was washed with 10% citric acid, 5% Na₂CO₃ and water, dried overNa₂SO₄ and evaporated down. Petroleum ether was added to the residue toobtain a precipitate, which was collected by filtration. The crudeproduct in CHCl₃ (5 ml) was applied to a silica gel column (BW-127ZH,3×16 cm), which was equilibrated and eluted with CHCl₃ (2100 ml). Afterremoval of the solvent of the effluent (1,500 ml-2,100 ml), petroleumether was added to the residue to yield crystals, which were collectedby filtration, yield 300 mg (58.4%), mp 214-217° C., Rf¹ 0.46, Rf² 0.32.

[0058] Anal. Calcd for C₃₆H₅₄N₄O₈.0.5H₂O: C, 63.6; H, 8.15; N, 8.24.Found: 63.8; H, 8.16; N, 8.04.

2) 1,4-Bis-(Dmt-amino)butane.2HCl

[0059] A solution of 1,4-bis-(N^(α)-Boc-Dmt-amino)butane (200 mg, 0.30mmol) in TFA (1.0 ml, 13 mmol) containing anisole (0.10 ml, 0.90 mmol)was stirred for 1 hr at room temperature. Ether was added to a solutionto form a precipitate, which was collected by filtration and dried invacuo. The solution of the product in 1 mol/l HCl (0.6 ml) waslyophilized to yield a fluffy powder, yield 110 mg (68%), Rf⁴ 0.19, Rf⁵0.32, tR (C) 14.97 min. TOF-MS m/z: 472.0 (M+1)⁺ (Calcd for C₂₆H₃₈N₄O₄:470.6).

Example 2 1) 1-Boc-amino-4-(N^(α)-Fmoc-Phe-amino)butane

[0060] 1-(Boc-amino)-4-aminobutane (2.0 g, 10 mmol), N^(α)-Fmoc-Phe-OH(4.6 g, 12 mmol), PyBOP (6.24 g, 10 mmol) and HOBt (1.6 g, 12 mmol) weredissolved in DMF (50 ml) containing DIEA (4.2 ml, 24 mmol). The reactionmixture was stirred for 15 hr at room temperature. After removal of thesolvent, AcOEt was added to the residue to obtain a precipitate, whichwas collected by filtration and recrystallized from EtOH, yield 3.0 g(45%), mp 165-167° C., Rf¹ 0.66.

[0061] Anal. Calcd for C₃₃H₃₉N₃O₅.0.25H₂O: C, 70.5; H, 7.08; N, 7.47.Found: C, 70.5; H, 7.06; N, 7.64.

2) 1-N^(α)-(Boc-Dmt-amino-4-(N^(α)-Fmoc-Phe-amino)butane

[0062] N^(α)-Boc-Dmt-OH (460 mg, 1.50 mmol),1-amino-4-(N^(α)-Fmoc-Phe-amino)butane.TFA [prepared from1-Boc-amino-4-(N^(α)-Fmoc-Phe-amino)butane (830 mg, 1.50 mmol), anisole(0.25 ml, 2.3 mmol) and TFA (2.2 ml, 30 mmol) as usual], PyBOP (930 mg,1.8 mmol) and HOBt (270 mg, 1.8 mmol) were dissolved in DMF (10 ml)containing DIEA (0.50 ml, 3.80 mmol). The reaction mixture was stirredfor 15 hr at room temperature. After removal of the solvent, the residuewas extracted with AcOEt, which was washed with 10% citric acid, 5%Na₂CO₃ and water, dried over Na₂SO₄ and evaporated down. Petroleum etherwas added to the residue to yield a precipitate. The crude product inCHCl₃ (5 ml) was applied to a silica gel column (YMC 70-230 mesh, 3×16cm), which was equilibrated and eluted with CHCl₃. After removal of thesolvent of the effluent (210 ml), petroleum ether was added to theresidue to give crystals, which were collected by filtration, yield 600mg (63%), mp 201-203.5° C., Rf¹ 0.52.

[0063] Anal. Calcd for C₄₄H₅₂N₄O₇.0.3H₂O: C, 70.0; H, 7.03; N, 7.42.Found: C, 70.0; H, 7.44; N, 7.48.

3) 1-Dmt-amino-4-Phe-aminobutane.2HCl

[0064] 1-(N^(α)-Boc-Dmt-amino)-4-(N^(α)-Fmoc-Phe-amino)butane (500 mg,0.78 mmol) was treated with 20% piperidine in DMF (11.5 ml) for 2 hr atroom temperature. After removal of the solvent, ether was added to theresidue to obtain a precipitate, which was collected by filtration (Rf¹0.16, Rf³ 0.70). This product (300 mg, 0.57 mmol) was dissolved in TFA(1.0 ml, 13 mmol) containing anisole (0.10 ml, 0.90 mmol) and thesolution was stirred for 1 hr at room temperature. Ether was added tothe solution to give a precipitate, which was collected by filtration.The crude product was purified with HPLC and lyophilized from 1 mol/lHCl to obtain fluffy amorphous powder, yield 220 mg (56%), Rf⁴ 0.30, Rf⁵0.46, tR(C) 17.07 min. TOF-MS m/z: 427.5 (M+1)⁺ (Calcd for C₂₄H₃₄N₄O₃:426.6).

[0065]¹H-NMR (free compound, DMSO-d6) δ: 8.648 (1H, t, J=5.5 HZ, NH ofPhe amide), 7.997 (1H, t, J=5.5, NH of Dmt amide), 7.32-7.23 (5H, m,aromatic H of Phe), 6.441 (2H, s, aromatic H of Dmt), 4.043 (1H, t,J=7.0, α-H of Phe), 3.711 (1H, dd, J=9.3, 6.4, α-H of Dmt), 3.071 (2H,d, J=7.0, β-CH₂ of Phe), 3.01-2.70 (6H, m, 1,4-CH₂+β-CH₂ of Dmt), 2.187(6H, s, diMe of Dmt), 1.16-1.01 (4H, m, 2,3-CH₂), 13C-NMR (DMSO-d6) δ:167.91 (q, >C═O of Dmt), 167.52 (q, >C═O of Tyr), 155.54 (q, >C═, 4 ofDmt), 138.14 (q, >C═, 2,6 of Dmt), 135.10 (t, >C═, 1 of Phe), 129.42 (t,H—C═, 2,6 of Phe), 128.25 (t, H—C═, 3,5 of Phe), 126.84 (t, H—C═, 4 ofPhe), 122.08 (q, >C═, 1 of Dmt), 114.80 (t, H—C═, 3,5 of Dmt), 53.39 (t,H—C<, α of Phe), 51.65 (t, H—C<, α of Dmt), 38.09 (s, —CH₂—, 1 or 4CH₂), 38.05 (s, —CH₂—, 4 or 1 CH₂), 36.83 (s, β of Phe), 30.44 (s, β ofDmt), 25.52 (s, —CH₂—, 2 or 3 CH₂), 25.48 (s, —CH₂—, 3 or 2 CH₂), 19.90(p, —CH₃, diMe of Dmt).

Example 3 1) 1-Boc-amino-4-N^(α)-Fmoc-Tyr(BrZ)-aminobutane

[0066] 1-Boc-amino-4-aminobutane (0.94 g, 5 mmol),N^(α)-Fmoc-Tyr(BrZ)-OH (3.1 g, 12 mmol), and PyBOP (6.24 g, 12 mmol)were dissolved in DMF (30 ml) containing DIEA (1.7 ml, 10 mmol). Thereaction mixture was stirred for 15 hr at room temperature. Afterremoval of the solvent, AcOEt and 5% Na₂CO₃ were added to the residue togive crystals, which were collected by filtration and recrystallizedfrom EtOH, yield 3.1 g (80%), mp 145-148° C., Rf¹ 0.80, Rf² 0.80.

[0067] Anal. Calcd for C₄₁H₄₄N₃O₈Br: C, 62.6; H, 5.63; N, 5.34. Found:C, 62.6; H, 5.65; N, 5.25.

2) 1-(N^(α)-Boc-Dmt-amino)-4-N^(α)-Fmoc-Tyr(BrZ)-aminobutane

[0068] N^(α)-Boc-Dmt-OH (550 mg, 1.80 mmol),1-amono-4-[N^(α)-Fmoc-Tyr(BrZ)]-aminobutane.TFA [prepared fromBoc-amino-4-[N^(α)-Fmoc-Tyr(BrZ)]-aminobutane (1.2 g, 1.5 mmol), anisole(0.25 ml, 2.3 mmol) and TFA (2.2 ml, 30 mmol) as usual], and PyBoP (1.12g, 2.2 mmol) were dissolved in DMF (20 ml) containing DIEA (0.88 ml, 5.1mmol). The reaction mixture was stirred for 15 hr at room temperature.After removal of the solvent, the residue was extracted with AcOEt,which was washed with 10% citric acid, 5% Na₂CO₃ and water, dried overNa₂SO₄ and evaporated down. Ether was added to the residue to yieldcrystals, which were collected by filtration and recrystallized fromEtOH, yield 1.1 g (63%), mp 186-190° C., Rf¹ 0.77.

[0069] Anal Calcd for C₅₂H₅₇N₄O₁₀Br: C, 63.9; H, 5.87; N, 5.72. Found:C, 63.6; N, 5.84; N, 5.50.

3) 1-(Dmt-amino)-4-Tyr-aminobutane.2HCl

[0070] 1-N^(α)-Boc-Dmt-amino-4-N^(α)-Fmoc-Tyr(BrZ)-aminobutane (600 mg,0.61 mmol) was treated with 20% piperidine in DMF (15 ml) for 2 hr atroom temperature. After removal of the solvent, ether was added to theresidue to obtain a precipitate, which was collected by filtration (Rf¹0.04, Rf³ 0.57). A solution of the resulting product (220 mg, 0.41 mmol)in TFA (1.0 ml, 13 mmol) was stirred for 1 hr at room temperature. Etherwas added to the solution to give a precipitate, which was collected byfiltration, dried in vacuo and purified with HPLC. The purified productwas lyophilized from 1 mol/l HCl to yield a white fluffy amorphouspowder, yield 100 mg (32%), Rf⁴ 0.20, RF⁵ 0.36, tR (C) 15.78 min. TOF-MSm/z: 443.8 (M+1)⁺ (Calcd for C₂₄H₃₄N₄O₄: 442.5).

[0071]¹H-NMR (free compound, DMSO-d6) δ: 8.541 (1H, t, J=5.4 HZ, NH ofTyr amide), 7.972 (1H, t, J=5.4, NH of Dmt amide), 7.046 (2H, d-like,J=8.5, 2,6 H of Tyr), 6.717 (2H, d-like, J=8.5, 3,5 H of Tyr), 6.434(2H, s,3,5 H of Dmt), 3.923 (1H, t, J=7.0, α-H of Tyr), 3.701 (1H, dd,J=10.6, α-H of Dmt), 3.02-2.79 (8H, m, 1,4-CH₂+β-CH₂ of Tyr and Dmt),2.184 (6H, s, diMe of Dmt), 1.18-1.03 (4H, m, 2,3-CH₂), ¹³C-NMR(DMSO-d6) δ: 167.91 (q, >C═O of Dmt), 167.63 (q, >C═O of Tyr), 156.40(q, >C═, 4 of Tyr), 155.52 (q, >C═, 4 of Dmt), 138.16 (q, >C═, 2,6 ofDmt), 130.31 (t, H—C═, 2,6 of Tyr), 124.89 (q, >C═, 1 of Tyr), 122.08(q, 1 of Dmt), 115.15 (t, H—C═, 3,5 of Tyr), 114.81 (t, H—C═, 3,5 ofDmt), 53.69 (t, H—C<, α of Tyr), 51.67 (t, H—C<, α of Dmt), 38.11 (s,—CH₂—, 1 or 4 CH₂), 38.05 (s, —CH₂—, 4 or 1 CH₂), 36.12 (s, β of Tyr),30.45 (s, β of Dmt), 25.56 (s, —CH₂—, 2 or 3 CH₂), 25.52 (s, —CH₂—, 3 or2 CH₂), 19.88 (p, —CH₃, diMe of Dmt).

Example 4 1) 1,6-Bis(N^(α)-Boc-Dmt-amino)hexane

[0072] N^(α)-Boc-Dmt-OH (500 mg, 1.6 mmol), 1,6-diaminohexane (92 mg,0.80 mmol) and PyBOP (1.0 g, 1.9 mmol) were dissolved in DMF (15 ml)containing Et₃N (0.27 ml, 1.9 mmol). The reaction mixture was stirredfor 18 hr at room temperature. After removal of the solvent, the residuewas extracted with AcOEt, which was washed with 10% citric acid, 5%Na₂CO₃ and water, dried over Na₂SO₄ and evaporated down. Petroleum etherwas added to the residue to give a precipitate, which was collected byfiltration. The crude product in AcOEt-n-hexane (1:1, 5 ml) was appliedto a silica gel column(BW-127ZH, 3×16 cm), equilibrated and eluted withAcOEt-n-hexane (1:1, 300 ml). After removal of the solvent of theeffluent (1-200 ml), petroleum ether was added to the residue to form aprecipitate, which was collected by filtration, yield 300 mg (53%), mp183-186° C., Rf¹ 0.52, Rf² 0.08.

[0073] Anal. Calcd for C₃₈H₅₈N₄O₈.0.25H₂O: C, 64.9; H, 8.37; N, 7.96.Found: C, 65.0; H, 8.22; N, 7.78.

2) 1,6-Bis(Dmt-amino)hexane.2HCl

[0074] A solution of 1,6-bis(N^(α)-Boc-Dmt-amino)hexane (150 mg, 0.21mmol) and anisole (0.10 ml, 0.90 mmol) in TFA (1.0 ml, 13 mmol) wasstirred for 1 hr at room temperature. Ether was added to the solution toform a precipitate, which was collected by filtration, dried in vacuoand lyophilized from 1 mol/l HCl, yield 90 mg (75%), Rf⁴ 0.21, Rf⁵ 0.42,tR (C) 18.15 min. TOF-MS m/z: 499.2 (M+1)⁺ (Calcd for C₂₈H₄₂N₄O₄:498.6).

Example 5 1) 1,2-Bis(N^(α)-Boc-Dmt-amino)ethane

[0075] N^(α)-Boc-Dmt-OH (500 mg, 1.6 mmol), 1,2-diaminoethane (50 μl,0.80 mmol), BOP (840 mg, 1.9 mmol) and HOBt (260 mg, 1.9 mmol) weredissolved in DMF (15 ml) containing Et₃N (0.54 ml, 3.8 mmol). Thereaction mixture was stirred for 18 hr at room temperature. Afterremoval of the solvent, the residue was extracted with AcOEt, which waswashed with 10% citric acid, 5% Na₂CO₃, and water, dried over Na₂SO₄ andevaporated down. Petroleum ether was added to the residue to yieldcrystals, which were collected by filtration and recrystallized fromEtOH, yield 300 mg (58%), mp 201-205° C., Rf¹ 0.48.

[0076] Anal. Calcd for C₃₄H₅₀N₄O₈.0.5H₂O: C, 62.7; H, 7.88; N, 8.59.Found: C, 62.5; H, 7.80; N, 8.30.

2) 1,2-Bis(Dmt-amino)ethane.2TFA

[0077] A solution of 1,2-bis(N^(α)-Boc-Dmt-amino)ethane (200 mg, 0.30mmol) and anisole (0.1 ml, 0.90 mmol) in TFA (1.0 ml, 13 mmol) wasstirred for 1 hr at room temperature. Ether was added to the solution toform a precipitate, which was collected by filtration, dried in vacuoand lyophilized from water, yield 120 mg (60%), Rf⁴ 0.14, Rf⁵ 0.58, tR(C) 13.8 min. TOF-MS m/z: 443.6 (M+1)⁺ (Calcd for C₂₄H₃₄N₄O₄: 442.5).

Example 6 1) 1,8-Bis(N^(α)-Boc-Dmt-amino)octane

[0078] N^(α)-Boc-Dmt-OH (500 mg, 1.6 mmol), 1,8-diaminooctane (120 mg,0.80 mmol), BOP (840 mg, 1.9 mmol) and HOBt (260 mg, 1.9 mmol) weredissolved in DMF (15 ml) containing Et₃N (0.54 ml, 3.8 mmol). Thereaction mixture was stirred for 18 hr at room temperature. Afterremoval of the solvent, the residue was extracted with AcOEt, which waswashed with 10% citric acid, 5% Na₂CO₃ and water, dried over Na₂SO₄ andconcentrated. Petroleum ether was added to the residue to give crystals,which were collected by filtration and recystallized from AcOEt, yield310 mg (53%), mp 142-144° C., Rf¹ 0.56.

[0079] Anal. Calcd for C₄₀H₆₂N₄O₈.0.5H₂O: C, 65.3; 8.62; N, 7.61. Found:C, 65.2; H, 8.40; N, 7.68.

2) 1,8-Bis(Dmt-amino)octane.2TFA

[0080] A solution of 1,8-bis(N^(α)-Boc-Dmt-amino)octane (260 mg, 1.6mmol) and anisole (0.1 ml, 0.90 mmol) in TFA (1.0 ml, 13 mmol) wasstirred for 1 hr at room temperature. Ether was added to the solution toform a precipitate, which was collected by filtration, dried in vacuoand lyophilized from water, yield 120 mg (60%), Rf⁴ 0.56, Rf⁵ 0.68, tR(C) 20.7 min. TOF-MS m/z: 527.7 (M+1)⁺ (Calcd for C₂₄H₃₄N₄O₄: 526.7).

Reference Example 1 1) 1,4-Bis(N^(α)-Boc-Phe-amino)butane

[0081] N^(α)-Boc-Phe-OH (1.1 g, 4.0 mmol), 1,4-diaminobutane (180 mg,2.0 mmol) and PyBOP (2.5 g, 4.8 mmol) were dissolved in DMF (25 ml)containing Et₃N (0.67 ml, 4.8 mmol). The reaction mixture was stirredfor 15 hr at room temperature. After removal of the solvent, AcOEt andwater were added to the residue to obtain crystals, which were collectedby filtration and recrystallized from EtOH, yield 0.75 g (32%), mp186-189° C., Rf¹ 0.65, Rf² 0.45.

[0082] Anal. Calcd for C₃₂H₄₆N₄O₆: C, 66.0; H, 7.96; N, 9.62. Found: C,65.9; H, 7.75; N, 9.61.

2) 1,4-Bis(Phe-amino)butane.2HCl

[0083] A solution of 1,4-bis(N^(α)-Boc-Phe-amino)butane (580 mg, 1.0mmol) and anisole (0.15 ml, 1.4 mmol) in TFA (1.5 ml, 20 mmol) wasstirred for 1 hr at room temperature. Ether was added to the solution toform a precipitate, which was collected by filtration. The crude productin 3% AcOH (3 ml) was applied to a Sephadex G-15 column (2.5×43 cm),equilibrated and eluted with 3% AcOH. Fractions (8 g each) werecollected and the solvent of the effluent (Nos. 8-11) was removed. Theresidue was lyophilized from 1 mol/l HCl to yield an amorphous powder,yield 340 mg (75%), Rf⁴ 0.27, Rf⁵ 0.47, tR (C) 21.04 min. TOF-MS m/z:383.3 (M+1)⁺ (Calcd for C₂₂H₃₀N₄O₂: 382.4).

Reference Example 2 1) 1,4-Bis(N^(α)-Boc-Tyr-amino)butane

[0084] N^(α)-Boc-Tyr-OH (5.6 g, 20 mmol), 1,4-diaminobutane (880 mg, 10mmol), BOP (10.6 g, 24 mmol) and HOBt (3.0 g, 20 mmol) were dissolved inDMF (50 ml) containing Et₃N (5.6 ml. 40 mmol). The reaction mixture wasstirred for 15 hr at room temperature. After removal of the solvent, theresidue was extracted with AcOEt, which was washed with 5% Na₂CO₃ andwater, dried over Na₂SC₄ and evaporated down. Ether was added to theresidue to form a precipitate, which was collected by filtration, yield4.2 g (68%), mp 123-126° C., Rf¹ 0.40.

[0085] Anal. Calcd for C₃₂H₄₆N₄O₈.0.7H₂O: C, 61.3; H, 7.56; N, 8.93.Found: C, 61.4. H, 7.81; N, 8.78.

2) 1,4-Bis(Tyr-amino)butane.2HCl

[0086] A solution of 1,4-bis(N^(α)-Boc-Tyr-amino)butane (550 mg, 0.90mmol) and anisole (0.28 ml, 2.6 mmol) in TFA (2.75 ml, 36 mmol) wasstirred for 1 hr at room temperature. Ether was added to the solution toform a precipitate, which was collected by filtration and lyophilizedfrom 1 mol/l HCl yield 340 mg (70%), Rf⁴ 0.27, tR (C) 7.1 min. TOF-MSm/z: 414.1 M⁺ (Calcd for C₂₂H₃₀N₄O₄: 414.4).

Reference Example 3 1) 1,6-Bis(N^(α)-Boc-Tyr-amino)hexane

[0087] N^(α)-Boc-Tyr-OH (5.6 g, 20 mmol), 1,6-diaminohexane (1.9 g, 10mmol), BOP (11 g, 24 mmol), and HOBt (3.0 g, 20 mmol) were dissolved inDMF (50 ml) containing Et₃N (5.6 ml, 40 mmol). The reaction mixture wasstirred for 15 hr at room temperature. After removal of the solvent, theresidue was extracted with AcOEt, which was washed with 5% Na₂CO₃ andwater, dried over Na₂SO₄ and evaporated down. Ether was added to theresidue to form a precipitate, which was collected by filtration, yield5.0 g (79%), mp 146-151° C., Rf¹ 0.56.

[0088] Anal. Calcd for C₃₄H₅₀N₄O₈.2.5H₂O: C, 59.4; H, 8.06; N, 8.15.Found: C, 59.2; H, 7.84; N, 8.11.

2) 1,6-Bis(Tyr-amino)hexane.2TFA

[0089] A solution of 1,6-bis(N^(α)-Boc-Tyr-amino)hexane (500 mg, 1.6mmol) and anisole (0.12 ml, 1.1 mmol) in TFA (1.2 ml, 16 mmol) wasstirred for 1 hr at room temperature. Ether was added to the solution toform a precipitate, which was collected by filtration, dried in vacuoand lyophilized from water, yield 250 mg (48%), Rf⁴ 0.31, tR (Y) 20.1min TOF-MS m/z: 443.1 M⁺ (Calcd for C₂₂H₃₀N₄O₄:442.5).

Reference Example 4 1) 1,2-Bis(N^(α)-Boc-Tyr-amino)ethane

[0090] N^(α)-Boc-Tyr-OH (4.1 g, 15 mmol), 1,2-diaminoethane (0.36 g, 6.0mmol) and BOP (7.6 g, 17 mmol) were dissolved in DMF (40 ml) containingEt₃N (2.4 ml, 17 mmol). The reaction mixture was stirred for 15 hr atroom temperature. After removal of the solvent, the residue wasextracted with AcOEt, which was washed with 5% Na₂CO₃ and water, driedover Na₂SO₄ and concentrated. Petroleum ether was added to the residueto form a precipitate. The crude product in CHCl₃ (5 ml) was applied toa silica gel column (BW-127ZH, 3×34 cm), equilibrated and eluted withCHCl₃. After removal of the solvent of the effluent (300-2,300 ml),petroleum ether was added to the residue to obtain a precipitate, yield1.0 g (30%), mp 203-206° C., Rf¹ 0.49.

[0091] Anal. Calcd for C₃₀H₄₂N₄O₈.0.7H₂O: C, 60.1; H, 7.29; N, 9.34.Found: C, 60.2; H, 6.96; N, 9.03.

2) 1,2-Bis(Tyr-amino)ethane.2TFA

[0092] A solution of 1,2-bis(N^(α)-Boc-Tyr-amino)ethane (500 mg, 0.90mmol) and anisole (0.14 ml, 1.3 mmol) in TFA (1.4 ml, 18 mmol) wasstirred for 1 hr at room temperature. Ether was added to the solution toform a precipitate. The precipitate was purified with HPLC andlyophilized from water, yield 350 mg (63%), Rf⁴ 0.30, tR (C) 5.5 min.TOF-MS m/z: 387.0 (M+H)⁺ (Calcd for C₂₀H₂₆N₄O₄: 386.4).

Reference Example 5 1) 1,8-Bis(N^(α)-Boc-Tyr-amino)octane

[0093] N^(α)-Boc-Tyr-OH (5.6 g, 20 mmol), 1,8-diaminooctane (1.4 g, 10mmol) and BOP (11 g, 24 mmol) were dissolved in DMF (50 ml) containingEt₃N (3.4 ml, 24 mmol). The reaction mixture was stirred for 15 hr atroom temperature. After removal of the solvent, the residue wasextracted with AcOEt, which was washed with 5% Na₂CO₃ and water, driedover Na₂SO₄ and concentrated. Petroleum ether was added to the residueto form a precipitate, which was collected by filtration, yield 6.1 g(91%), mp 159-160° C., Rf⁴ 0.60.

[0094] Anal. Calcd for C₃₆H₅₄N₄O₈.0.5H₂O: C, 63.6; H, 8.09; N, 8.23.Found: C, 63.9; H, 8.23; N, 7.97.

2) 1,8-Bis(Tyr-amino)octane.2TFA

[0095] A solution of 1,8-bis(N^(α)-Boc-Tyr-amino)octane (1.0 g, 1.5mmol) and anisole (0.45 ml, 4.2 mmol) in TFA 4.5 ml, 60 mmol) wasstirred for 1 hr at room temperature. Ether was added to the solution toform a precipitate, which was purified with HPLC and lyophilized fromwater, yield 320 mg (30%), Rf⁴ 0.30, tR (C) 18.3 min. TOF-MS m/z: 471.2(M+1)⁺ (Calcd for C₂₆H₃₈N₄O₄: 470.4).

Reference Example 6 Boc-Dmt-OH

[0096] 2,6-Dimethyl-L-tyrosine mono hydrochloride (4.7 g, 0.018 mol) wasdissolved in water and thereto was added triethylamine (4.9 ml, 0.036mol). Di-t-butyldicarbonate (3.9 g, 0.020 mol) in dioxane (20 ml) wasadded to the above solution. The solution was stirred overnight at roomtemperature. After the reaction, the solvent was removed and theresidual oil was suspended in citric acid solution. The suspension wasextracted with AcOEt. The organic layer was washed with saturated brineand water and then dried over sodium sulfate. The solvent was removedand to the residue was added hexane to form a precipitate. Theprecipitate was collected by suction to give Boc-Dmt-OH (yield 5.5 g).mp 174-177° C., Rf⁴ 0.80, [α]_(D) ²⁵=−11.0(C=1, MeOH)

[0097] Anal. Calcd for C₁₆H₂₃NO₅.0.1H₂O: C, 61.8; H, 7.51; N, 4.50.Found: C, 61.8; H, 7.54; N, 4.45.

[0098] Regarding the opioid peptide derivatives of the presentinvention, as shown in the following Experimental Examples, the opioidreceptor affinity was measured by performing a receptor competitiveassay using mouse brain-derived opioid receptors, whether the derivativeis an agonist or an antagonist was tested by a guinea pig ileum (GPI)method and a mouse seminiferous tract (MVD) method, and finally theanalgesic effect was measured by a Tail Pressure method and, whereby,the pharmacological effects of the present opioid peptide derivativeswere confirmed.

Experimental Example 1

[0099] Measurement of the Opioid Receptor Affinity:

[0100] Regarding the opioid peptide derivatives synthesized in Examplesand Reference Examples, the μ- and δ-opioid receptor affinities wereobtained by a competitive receptor assay using the synaptic membranefraction obtained from the rat brain tissue according to the knownmethods. That is, Sprague-Dawley rats having an average weight of150-180 g were killed by decapitation, the brain tissue from whichcerebellum had been removed was homogenized in 0.32M sucrose and 1 mMHEPES (pH 7.5) containing 50 μg/ml soybean trypsin inhibitor,fractionated and centrifuged to obtain the synaptic membrane fraction(P2 fraction), which was pre-incubated in 50 mM HEPES (pH 7.5)containing 100 mM NaCl, 0.1 mM GDP and soybean trypsin inhibitor toremove endogenous ligands from the P2 fraction.

[0101] A competitive receptor binding assay (Radiolabeled receptorassay: RRA) based on an exchange reaction between a radioactive ligandfor the synaptic membrane fraction and a peptide derivative to be testedwas performed by using tritium-labeled [³H]DAGO which is a μ-opioidreceptor agonist and tritium-labeled [³H]DPDPE which has the highδ-opioid receptor selectivity, and incubating an aliquot of the synapticmembrane with the peptide derivative to be tested for a period of time.Upon this, an exchange reaction between a ligand was performed underconstant conditions, the radiation remaining in the synaptic membranefraction was measured, and an amount of the ligand adsorbed onto thesynaptic membrane fraction is calculated. By assuming that binding witha receptor occurs in competition between a radioactive ligand and an endligand, the concentration (IC₅₀) at which the maximum specific bindingof the radioactive ligand is 50% inhibited was obtained, from which theaffinity constant (Ki) was calculated by a method of Cheng et al.(Biochem. Pharmcol., 22, 3099(1973)).

[0102] The results are shown in the following Table. TABLE 1 Affinityand selectivity of compounds on μ- and δ- receptors Affinity andselectivity of receptor (Ki, nM) Compound δ μ δ/μ Ref.ex.2: TyrN 6,500 ±278   309 ± 104 21 H—(CH₂)₄—NHTyr Ref.ex.1: PheN 46,200 ± 458   1,530 ±122   30 H—(CH₂)₄—NHPhe Ex.2: DmtNH— 61.0 ± 5.7   0.52 ± 0.089 117(CH₂)₄—NHPhe Ex.3: DmtNH— 133 ± 18  0.38 ± 0.02 349 (CH₂)₄—NHTyr Ex.1:DmtNH— 53.4 ± 14.8 0.041 ± 0.003 1300 (CH₂)₄—NHDmt Ref.ex.4: TyrN 8,290± 43.3  648 ± 95  13 H—(CH₂)₂—NHTyr Ex.5: DmtNH—  116 ± 10.6 1.43 ± 0.0181 (CH₂)₂—NHDmt Ref.ex.3: TyrN 21,900 ± 4,020  410 ± 85  53H—(CH₂)₆—NHTyr Ex.4: DmtNH— 46.1 ± 8.8  0.053 ± 0.01  870 (CH₂)₆—NHDmtRef.ex.5: TyrN 6,150 ± 525    399 ± 40.5 34 H—(CH₂)₈—NHTyr Ex.6: DmtNH—14.8 ± 3.0   0.19 ± 0.024 78 (CH₂)₈—NHDmt

[0103] It was confirmed that compounds of the present invention have theaffinity for a μ-receptor at the very low concentration.

Experimental Example 2

[0104] Measurement of the agonist and antagonist activities on variousopioid receptors:

[0105] An assay test for agonist and antagonist activities of opioidcompounds was performed using guinea pig ileum (GPI) and mouseseminiferous tract (MVD). That is, male guinea pigs having the weight ofaround 300 g were killed by exsanguination, an ileum section having alength of around 10 cm was isolated from an ileum close to anileum-caecum region was held at 36° C. The ileum section was placed in aMagnus tube treated with an oxygen mixture (95% O₂/5% CO₂) and filledwith Krebs solution in the state where the rest tension of 1 g wasapplied with an isotonic transducer and, then, was performed. Then, theelectrical stimulation was applied at 30V for 0.5 ms. via electrodepreviously installed in the Magnus tube to constrict guinea pig ileumlongitudinal muscle and, after constriction was stabilized, a peptidaseinhibitor was added. Then, the present peptide derivative to be testedwas added, the constriction of guinea pig ileum longitudinal muscle wastaken by GrassFTO 0.3 transducer and monitored by an amplifier and arecorder connected to the transducer. The opioid activity was determinedby constriction inhibition after addition of the peptide derivative tobe tested and constriction inhibition cancellation by addition ofnaloxone, a selective opioid antagonist. The concentration at which theconstriction by electrical stimulation of guinea pig ileum longitudinalmuscle is 50% inhibited was measured, which was used as IC₅₀.

[0106] In addition, in a mouse MVD assay, the similar procedures werecarried out using mouse seminiferous tract in place of guinea pig ileum,and the concentration IC₅₀ at which the constriction by electricalstimulation of mouse seminiferous tract is 50% inhibited was determined.

[0107] Since it was made clear that the opioid activity is mediatedmainly by μ-opioid receptor in a GPI assay, whereas inhibition of theelectrical constriction is by the interaction with δ-opioid receptor,the concentration at which each constriction by electrical stimulationwas 50% inhibited was regarded as the agonist effect.

[0108] The results obtained on the present peptide derivatives are shownin following Table. TABLE 2 Agonist and antagonist activities ofcompounds on μ- and δ- receptors Agonist and antagonist activities (nM)Compound GPI(IC₅₀ ± S.E.) MVD(IC₅₀ ± S.E.) Ex.2: DmtNH— 181 ± 36  >10000ant. (5.5) (CH₂)₄—NHPhe Ex.3: DmtNH— 225 ± 11  >10000 ant. (5.3)(CH₂)₄—NHTyr Ex.1: DmtNH— 5.33 ± 0.65 >10000 ant. (5.8) (CH₂)₄—NHDmtEx.5: DmtNH— 2840 ± 517  >10000 ant. (5.5) (CH₂)₂—NHDmt Ex.4: DmtNH—3.08 ± 0.53 >10000 ant. (6.1) (CH₂)₆—NHDmt Ex.6: DmtNH— 53.7 ±7   >10000 ant. (6.4) (CH₂)₈—NHDmt

[0109] From these results, it can be seen that the present peptidederivatives are μ-receptor agonists.

[0110] The analgesic effects of1,4-bis(2,6-dimethyl-L-tyrosylamino)butane (Ex. 1) and1,6-bis(2,6-dimethyl-L-tyrosylamino)hexane (Ex. 4):

[0111] These compounds are novel compounds synthesized by the presentinventors. These compounds have been shown to have the high μ receptorbinding properties from a binding experiment to an opioid receptor of asynaptic membrane fraction. In addition, these compounds are recognizedto have the agonist activity to guinea ileum in an experiment using asection of guinea pig gut tract. Therefore, it is presumed that thesecompounds have the analgesic effects. Then, these compounds wereadministered intracerebroventricularly (i.v.c.), intravenously orsubcutaneonsly into a rat, and the analgesic effects were examined.

[0112] Method;

[0113] SD Male rats, weighing 110 to 130 g, were used. One groupconsisted of five rats. The analgesic effects were examined by aRandoll-Seritto method. That is, the pressure was applied to a hind limbof a rat using a pressure-stimulated analgesic effect measuringapparatus, and the threshold at which a rat exhibits an escape reactionwas measured, which was used as an index for the analgesic effect. Thethreshold was expressed in mm Hg. During the measurement, the upperlimit of the pressure applied to a hind limb was 100 mm Hg in order toavoid damage of the tissues.

[0114] 1) When performing intracerebroventricular administration(i.c.v.), a rat was fixed to a brain stereotactic fixing apparatus underslight ether anesthesia, the skin at a head was dissected, and anadministration cannula was inserted into a place at a transverse of 1.5to 2.0 nm from a bregma position and a depth of 3.5 to 4.0 mm. Theadministration volume was 10 μl/animal. Observation of the analgesicaction was performed before administration, and at 30 minutes, and 1, 2,3, 4 and 5 hours after administration. Doses of both compounds were 0.1,1 and 10 μg/animal. Separately, a group to which 5 mg/kg of naltrexonehydrochloride was administered subcutaneously immediately after compoundof Ex.1 10 μg/animal i.c.v and a group to which 5 mg/kg of naltrexonehydrochloride was administered subcutaneously immediately after compoundof Ex.4 10 μg/animal i.c.v. was made. In addition, as morphinehydrochloride at a dose of 3 μg/animal i.c.v. group was used as apositive control group and a physiological saline 10 μl/animal i.c.v.group as a control group was made, respectively.

[0115] 2) When administered intravenously (i.v.), a volume of 2 ml/kgweight was administered to a tail vein and, when administeredsubcutaneously (s.c.), a volume of 2 ml/kg weight was subcutaneouslyadministered to the back. In the case of intravenous administration, a 1mg/kg, 3 mg/kg or 10 mg/kg compound of Ex.1 administration group, a 1mg/kg, 3 mg/kg or 10 mg/kg compound of Ex.4 administration group, a 2ml/kg physiological saline administration group and a 5 mg/kg morphinehydrochloride administration group were made, respectively. In the caseof subcutaneous administration, a 10 mg/kg, 30 mg/kg or 100 mg/kgcompound of Ex.1 administration group, a 10 mg/kg, 30 mg/kg or 100 mg/kgcompound of Ex.4 administration group, a 2 ml/kg physiological salineadministration group and a 5 mg/kg morphine hydrochloride administrationgroup were made, respectively. Observation of the analgesic action wasperformed 15 minutes, 30 minutes, 1 hour and 2 hours afteradministration in the case of intravenous administration, 30 minutes, 1hour, 2 hours and 3 hours after administration in the case ofsubcutaneous administration, respectively.

[0116] In order to perform a significant difference test betweenrespective administration groups and control groups, and between valuesat respective times before administration and after administration, aDunnet multiple comparative test method was used. In the statisticalprocessing for performing a significant difference test between morphinehydrochloride administration groups and control groups, Student t-testwas used. For performing a significant difference test between values atrespective times before administration and after administration inmorphine hydrochloride administration groups, a Dunnet multiplecomparative test method was used. A significant level was at 5 or lower% risk rate (two-tailed test).

[0117] Results by intracerebroventricular administration are shown infollowing Table 3. TABLE 3 Effects in rats (intracerebroventricularly)Threshold (mmHg) Dose Time After administration (hr) Drugs (μg/animal,i.c.v.) Before 0.5 1 2 3 4 5 Control^(a)) — 47 ± 1 46 ± 1 46 ± 1 48 ± 145 ± 1 46 ± 1 46 ± 2 Ex. 1 0.1 47 ± 1 47 ± 1 50 ± 2 50 ± 1 48 ± 2 48 ± 149 ± 1 Ex. 1 1 47 ± 2 51 ± 3 54 ± 3 49 ± 1 49 ± 2 48 ± 2 47 ± 1 Ex. 1 1048 ± 2    61 ± 8*^(,$)    61 ± 5**^(,$) 54 ± 4 51 ± 3 52 ± 2  52 ± 1*Ex. 1 + 10 + 47 ± 2 49 ± 1 53 ± 3 54 ± 2 52 ± 2 49 ± 1 50 ± 1 Naltrexon5 mg/kg, s.c. Ex. 4 0.1 45 ± 2 47 ± 2 47 ± 1 50 ± 2 49 ± 2 49 ± 2 47 ± 1Ex. 4 1 46 ± 1 51 ± 2 51 ± 2 51 ± 3 50 ± 2    53 ± 2*^(,$)    52 ±1*^(,$) Ex. 4 10 46 ± 1 50 ± 2 47 ± 0 49 ± 2 48 ± 2 48 ± 2 49 ± 1 Ex.4 + 10 + 46 ± 1    55 ± 2*^(,$)    52 ± 2*^(,$) 51 ± 2 51 ± 1    52 ±1*^(,$) 50 ± 1 Naltrexon 5 mg/kg, s.c. Morphine 3 46 ± 1  59 ± 4^(#)  59± 3^(##) 52 ± 3  52 ± 3^(#) 49 ± 2 51 ± 2

[0118] In the physiological saline administration group, a significantchange was not seen in the escape reaction threshold at any time afteradministration. In the case where morphine hydrochloride 3 μg/animal wasadministered, a significant increase in the threshold was recognized 30minutes, 1 hour and 3 hours after administration as compared with beforeadministration.

[0119] In compound of Ex.1 administration group, a significant increasein the threshold was seen in the 10 μg/administration group 30 minutesand 1 hour after administration as compared with before administration.Increase in the threshold seen in compound of Ex.1 10 μg administrationgroup was dissipated by naltrexone 5 mg/kg subcutaneous administration.On the other hand, regarding compound of Ex.4, a significant increase inthe threshold was seen 4 hours and 5 hours after administration in the 1μg/animal administration group. However, in the 10 μg/animaladministration group, a significant change in the threshold was not seenat any time after administration. In the group where 5 mg/kg ofnaltrexone was administered subcutaneouly immediately afteradministration of 10 μg of compound of Ex.4, a significant increase inthe threshold was seen 30 minutes, 1 hour and 4 hours afteradministration.

[0120] Results by intravenous administration are shown in TABLE 4Effects in rats (intraveneous). Threshold (mmHg) Dose Time afteradministration (hr) Drugs (mg/kg, i.v.) Before 0.25 0.5 1 2 Control^(a))— 48 ± 2 48 ± 2 50 ± 2 51 ± 2 49 ± 2 Ex. 1 1 48 ± 2 50 ± 2 54 ± 2 53 ± 3 56 ± 2^($) Ex. 1 3 48 ± 2 55 ± 2 56 ± 3 52 ± 4 49 ± 2 Ex. 1 10 48 ± 2   60 ± 2**^(,$$) 56 ± 3 54 ± 2 53 ± 2 Ex. 1 1 48 ± 2 47 ± 2 51 ± 3 51 ±3 51 ± 3 Ex. 4 3 48 ± 2 57 ± 4 54 ± 3 54 ± 5 53 ± 3 Ex. 4 10 49 ± 2 54 ±3 53 ± 3 57 ± 2 52 ± 2 Morphine 5 49 ± 2    83 ± 6^(##,$$)    79 ±7^(##,$$)    78 ± 6^(##,$$) 65 ± 7

[0121] In the physiological saline administration group, a significantchange in the escape reaction threshold was not seen at any time afteradministration. In morphine hydrochloride 5 mg/kg administration group,a significant increase in the escape reaction threshold was recognized15 minutes, 30 minutes and 1 hour after administration as compared withbefore administration.

[0122] In compound of Ex.1 administration group, a significant increasein the threshold was seen 15 minutes after administration in the 10mg/kg administration group. In compound of Ex.4 administration group, asignificant change in the threshold was not seen at any measuring timeat any dose.

[0123] Results by subcutaneous administration are shown in followingTable 5. TABLE 5 Effects in rats (subcutaneous). Threshold (mmHg) DoseTime after administration (hr) Drugs (mg/kg, s.c.) Before 0.5 1 2 3Control^(a)) — 47 ± 2 49 ± 3 52 ± 2 48 ± 2 50 ± 3 Ex. 1 10 47 ± 2 55 ± 562 ± 6  58 ± 3* 57 ± 2 Ex. 1 30 47 ± 2 52 ± 4  65 ± 5^($$)    68 ±3**^(,$$) 59 ± 4 Ex. 1 100 47 ± 2    86 ± 2**^(,$$)    77 ± 2**^(,$$)   77 ± 2**^(,$$)    70 ± 3**^(,$$) Ex. 4 10 47 ± 2 58 ± 3 58 ± 5 55 ± 553 ± 1 Ex. 4 30 47 ± 2 59 ± 3 56 ± 3  65 ± 5^($$) 59 ± 4 Ex. 4 100 48 ±2    69 ± 6**^(,$$)    68 ± 3*^(,$)    75 ± 8**^(,$$) 58 ± 2 Morphine 548 ± 2    87 ± 2^(##,$$)    85 ± 6^(##,$$)   71 ± 7^(#,$) 61 ± 7

[0124] In the physiological saline administration group, a significantchange in the escape reaction threshold was not seen at any time afteradministration. In morphine hydrochloride 5 mg/kg administration group,a rapid increase in the threshold was seen and a significant increasewas recognized 30 minutes, 1 hour and 2 hours after administration ascompared with before administration. However, 3 hours afteradministration, a significant increase in the threshold was not seen.

[0125] In compound of Ex.1 10 mg/kg administration group, a tendency ofan increase in the threshold was seen. However, 2 hours afteradministration, only a significant difference between control groups wasseen 2 hours after administration. In the 30 mg/kg administration group,a significant increase in the threshold was seen 1 hour and 2 hoursafter administration as compared with before administration. In the 100mg/kg administration group, the similar increase in the threshold tothat in the case of morphine 5 mg/kg administration was seen, and asignificant increase was seen at any measuring time after administrationas compared with before administration.

[0126] In compound of Ex.4 10 mg/kg administration group, although atendency of a slight increase in the threshold was seen, a significantdifference was not seen before administration and between controlgroups. In the 30 mg/kg administration group, the threshold wasgradually increased, and a significant increase was seen 2 hours afteradministration as compared with the values before administration. In the100 mg/kg administration group, the threshold was increased rapidly, anda significant difference was seen in the measured values 30 minutes, 1hour and 2 hours after administration as compared with the values beforeadministration. However, 3 hours after administration, a significantincrease was not seen.

[0127] Also in the case of intravenous administration, compound of Ex.110 mg/kg exhibited an analgesic effect but is weaker as compared withthe case of morphine 10 mg/kg administration and its duration time wasshorter. On the other hand, no analgesic effect was seen in compound ofEx.4 at all doses used in the experiment.

[0128] When subcutaneously administered, both compound of Ex.1 andcompound of Ex.4 exhibited the analgesic effect. 10 mg/kg of compound ofEx.1 is weaker 2 hours after administration but exhibited thesignificant analgesic effect as compared with control groups. Thisanalgesic effect was exhibited earlier, became stronger and has a longerduration time by increasing the dose. The analgesic effect by compoundof Ex.1 100 mg/kg subcutaneous administration is approximatelycomparative to the effect by morphine 5 mg/kg subcutaneousadministration, and an effect duration time was longer than in morphine.Compound of Ex.4 30 mg/kg subcutaneous administration exhibited thesignificant analgesic effect 2 hours after administration. At the 100mg/kg administration, the significant analgesic effect was exhibitedfrom 30 minutes after administration. The analgesic effect of compoundof Ex.4 was weaker and has a shorter duration time than in compound ofEx.1. That it took a time before manifestation of the action and aduration time of the effect was prolonged with increasing doses shows apossibility that these compounds are absorbed comparatively slowly froma subcutaneous administration site or metabolites of these compound havethe analgesic action.

[0129] From the above experimental results, it was made clear thatcompound of Ex.1 and compound of Ex.4 have the analgesic effect via theμ-opioid receptor.

[0130] The peptide derivatives represented by the formula (1) and saltsthereof in accordance with the present invention are novel compounds notdescribed in the prior art, have the specific affinity for the opioidreceptor, and manifest a variety of morphine-like physiologicalactivities such as the analgesic effect.

[0131] Accordingly, the peptide derivatives (1) of the present inventionand salts thereof can be used as an analgesic drug or drugs for treatingor preventing nervous diseases associated with other opioid receptoractivities.

What is claimed is:
 1. A peptide derivative represented by the followingformula (1) or a salt thereof;

wherein R¹ is hydrogen atom or methyl group, R² is hydrogen atom orhydroxy group and n is an integer of 1 to 8, provided that R¹ ishydrogen atom when R² is hydrogen atom.
 2. The peptide derivativerepresented by the formula (1) or a salt thereof claimed in claim 1wherein R² is hydroxyl group.
 3. The peptide derivative represented bythe formula (1) or a salt thereof claimed in claim 1 wherein R¹ ismethyl group.
 4. The peptide derivative represented by the formula (1)or a salt thereof claimed in claim 1 wherein n is an integer of 4 to 6.5. A pharmaceutical composition containing a peptide derivativerepresented by the formula (1) or a salt thereof claimed in claim 1 asan active ingredient.
 6. A method for inhibiting or moderating paincomprising administering a peptide derivative represented by the formula(1) or a salt thereof claimed in claim 1 in an effective dosage to apatient having pain.
 7. A method for inhibiting or moderating the paincomprising administering a peptide derivative represented by the formula(1) or one salt thereof claimed in claim 1 in an effective dosage to apatient having pain or suffering from nervous defect disease related toμ-opioid receptor activation.